Coating haptens

Figure 13 Structures of haptens used for immunizing and coating antigens in a monoclonal antibody-based immunoassay for diuron. A sensitive assay was developed using coating hapten I that had the handle in a position different from the immunogen hapten. When the oxygen in the urea moiety of hapten I was replaced with a sulfur (hapten 11), increasing the heterology, even greater sensitivity was achieved...

Immobilization of A and B blood group oligosaccharide haptens and preparation of immunoadsorbents with specificity to anti-A and anti-B antibodies has been carried out with the use of poly acrylate-coated PG (WPG-PA) [124]. Prespacered A and B-trisaccharide-fl-aminopropylglycosides were used for the synthesis. WPG-PA (1 g) quantitatively binds both haptens (2 pinole) whereas some other activated affinity supports (for example, CNBr-Sepharose 4B) do not. On the other hand, glycidoxypropyl-silica binds prespacered haptens completely but these materials reveal no specific adsorptivity. 

The antibody solution (1.6x10 M) and substrate solutions with various concentration from 10 M to 10 M were mixed on a BSA-coated plate. The mixed solution of antibodies and substrates was allowed to stand for 1 day at room temperature, and then transported to the ELISA plates pre-coated with BSA-hapten and BSA blocking buffer. Absorbance at 405 nm for the resulting enzymatic hydrolysis product (p-nitrophenolate) by alkalinephosphatase of the second antibody was recorded on an Immuno-Mini NJ-2300 to determine the amount of antibody bound to BSA-hapten. 

Figure 1 Schematic of the quasi-equihbria using heterologous haptens in coating antigen immunoassay formats. Ka represents the equilibrium constant for binding of antibody (Y) to target analyte (A). Kh is the equilibrium constant for the binding of antibody to hapten-protein conjugate (H-) immobihzed on a solid phase...

Table 2 Guidelines for design of coating/tracer haptens...

Synthetic haptens mimicking some critical epitopic structures on larger macromolecules are often conjugated to carriers to create an immune response to the larger parent molecule. For instance, short peptide segments can be synthesized from the known sequence of a viral coat protein and coupled to a carrier to induce immunogenicity toward the native virus. This type of synthetic approach to immunogen production has become the basis of much of the current research into the creation of vaccines. 

HAp coatings, 14 105 HapMap project, 20 839 Hapten-antibody interactions, 9 64 Haptic sensor, for skin conditions, 3 749 Harcros antifoam, commercial defoamer, 3 24 It... 

We (K1) attempted to develop a noncompetitive assay based on the anti-idiotype antibodies for a conjugated bile acid metabolite, ursodeoxycholic acid 7-A-acetyl-glucosaminide (UDCA 7-NAG), which is expected to serve as a diagnostic index for an autoimmune disease, primary biliary cirrhosis. In our assay, the hapten UDCA 7-NAG, a /3-type antibody, and a biotin-labeled a-type antibody were simultaneously added to a microtiter plate coated with an F(ab )2 fragment of a specific anti-UDCA 7-NAG antibody, then incubated at room temperature for 8 h. Bound biotin was then detected with HRP-labeled streptavidin, whose enzyme activity was measured using o-phenylenediamine/H202 as a substrate. This noncompetitive assay system provided a subfemtomole-order sensitivity (detection limit 118 amol) that was 7 times lower than the competitive immunoassay using the same anti-hapten antibody (K2), even with a common colorimetric detection (Fig. 13). Somewhat improved specificity was also obtained namely, better... 

Fig. 10. Different techniques for competitive immuno-polymerase chain reaction of small-molecule compounds. (A) Using biotinylated haptens and a DNA-streptavidin nanocircle conjugate [105], hapten-DNA conjugates were synthesized and used for a competitive assay in a sample containing free hapten and capture antibody-coated surfaces [92]. (B) Hapten-coated microplates were simultaneously incubated with a sample containing free hapten and a hapten-specific antibody. Following competitive coupling, the immobilized antibody was subsequently detected by a species-specific antibody-DNA conjugate [93, 94].

In 1996, Gauglitz and coworkers coated surfaces with various amino-and carboxy-substituted polymers [198], The polymers tested were branched poly-(ethyleneimine), a,co-amino-functionalized PEG, chitosan, poly(acrylamide-co-acrylic acid) and an amino-modified dextran. The amino-substituted polymers were immobilized on glass by first immobilizing an aminosilane, followed by succinic anhydride/A-hydroxysuccinimide linker chemistry. Poly(acrylamide-co-acrylic acid) was directly coupled to an aminosilanized surface. When probed with 1 mg mL 1 ovalbumin solution, nonspecific adsorption was lowest for the dextran derivative. Notably, nonspecific adsorption increased in most cases when a hydrophobic hapten (atrazine) was coupled to the polymer-modified surface. 

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